Under strong sunlight, plants avoid photooxidation by quenching the excess absorbed energy. Quenching is triggered by PsbS, a membrane protein that is activated and deactivated by the light-dependent ...pH changes in the thylakoid lumen. The mechanism of action of this protein is unknown, but it was suggested that several glutamates act as pH sensors. However, the pK a of glutamate is several pH units below the physiological values in the lumen. Thus, how can PsbS sense the pH of the lumen, and how does it respond to it? By applying a nonstandard molecular dynamics method that treats pH explicitly, we show that the lumen-exposed glutamates of PsbS have strongly shifted pK a values and that such shifts are crucial for the pH sensitivity in physiological conditions. We also demonstrate that protonation drives a systematic unfolding of a region key for protein–protein interactions, indicating that PsbS response to pH is a functional conformational switch.
The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. ...Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide's effect on the membrane.
Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one ...of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286I, Y288I, and K362I. All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.
Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH₂) and N-terminal conjugation to ...ibuprofen (Ib), IbKTP-NH₂, was developed. The Ib moiety is an enhancer of KTP-NH₂ analgesic action. In the present study, we have tested the hypothesis that KTP-NH₂ is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH₂ conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH₂ and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH₂ tandem adopts a preferential “stretched” conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH₂ to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH₂. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH₂ given the enriched glycocalyx of the blood–brain barrier cells. Accumulation of IbKTP-NH₂ in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH₂ residue of IbKTP-NH₂ and free KTP-NH₂ in lipid membranes is the same.
The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to ...comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.
Ions are involved in multiple biological processes and may exist bound to biomolecules or may be associated with their surface. Although the presence of ions in nucleic acids has traditionally gained ...more interest, ion–protein interactions, often with a marked dependency on pH, are beginning to gather attention. Here we present a detailed analysis on the binding and distribution of ions around β-lactoglobulin using a constant-pH MD (CpHMD) method, at a pH range 3–8, and compare it with the more traditional Poisson–Boltzmann (PB) model and the existing experimental data. Most analyses used ion concentration maps built around the protein, obtained from either the CpHMD simulations or PB calculations. The requirements of approximate charge neutrality and ionic strength equal to bulk, imposed on the MD box, imply that the absolute value of the ion excess should be half the protein charge, which is in agreement with experimental observation on other proteins ( Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2015879118 ) and lends support to this protocol. In addition, the protein total charge (including territorially bound ions) estimated with MD is in excellent agreement with electrophoretic measurements. Overall, the CpHMD simulations show good agreement with the nonlinear form of the PB (NLPB) model but not with its linear form, which involves a theoretical inconsistency in the calculation of the concentration maps. In several analyses, the observed pH-dependent trends for the counterions and co-ions are those generally expected, and the ion concentration maps correctly converge to the bulk ionic strength as one moves away from the protein. Despite the overall similarity, the CpHMD and NLPB approaches show some discrepancies when analyzed in more detail, which may be related to an apparent overestimation of counterion excess and underestimation of co-ion exclusion by the NLPB model, particularly at short distances from the protein.
Protein–protein association is often mediated by electrostatic interactions and modulated by pH. However, experimental and computational studies have often overlooked the effect of association on the ...protonation state of the protein. In this work, we present a methodological approach based on constant-pH molecular dynamics (MD), which aims to provide a detailed description of a pH-dependent protein–protein association, and apply it to the dimerization of β-lactoglobulin (BLG). A selection of analyses is performed using the data generated by constant-pH MD simulations of monomeric and dimeric forms of bovine BLG, in the pH range 3–8. First, we estimate free energies of dimerization using a computationally inexpensive approach based on the Wyman–Tanford linkage theory, calculated in a new way through the use of thermodynamically based splines. The individual free energy contribution of each titratable site is also calculated, allowing for identification of relevant residues. Second, the correlations between the proton occupancies of pairs of sites are calculated (using the Pearson coefficient), and extensive networks of correlated sites are observed at acidic pH values, sometimes involving distant pairs. In general, strongly correlated sites are also slow proton exchangers and contribute significantly to the pH-dependency of the dimerization free energy. Third, we use ionic density as a fingerprint of protein charge distribution and observe electrostatic complementarity between the monomer faces that form the dimer interface, more markedly at the isoionic point (where maximum dimerization occurs) than at other pH values, which might contribute to guide the association. Finally, the pH-dependent dimerization modes are inspected using PCA, among other analyses, and two states are identified: a relaxed state at pH 4–8 (with the typical alignment of the crystallographic structure) and a compact state at pH 3–4 (with a tighter association and rotated alignment). This work shows that an approach based on constant-pH MD simulations can produce rich detailed pictures of pH-dependent protein associations, as illustrated for BLG dimerization.
Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We ...report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760-765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The pulmonary surfactant protein C (SP-C) is a small highly hydrophobic protein that adopts a mainly helical structure while associated with the membrane but misfolds into a β-rich metastable ...structure upon deacylation, membrane dissociation, and exposure to the neutral pH of the aqueous alveolar subphase, eventually leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. The present constant-pH MD study of the acylated and deacylated isoforms of SP-C in a chloroform/methanol/water mixture, often used to mimic the membrane environment, shows that the loss of the acyl groups has a structural destabilizing effect and that the increase of pH promotes intraprotein contacts which contribute to the loss of helical structure in solution. These contacts result from the poor solvation of charged groups by the solvent mixture, which exhibits a limited membrane-mimetic character. Although a single SP-C molecule was used in the simulations, we propose that analogous intermolecular interactions may play a role in the early stages of the protein misfolding and aggregation in this mixture.
Periodic boundary conditions (PBCs) are a standard feature of molecular simulations, and their mathematical and computational aspects are well-understood and relatively straightforward. However, they ...can in practice be a nuisance when simulating heterogeneous systems, especially when different types of molecules change their relative positions during the simulation. Although the translation required to fix a broken molecular complex of interest can in most cases be easily inferred by visual inspection, it typically depends on the type of system, its configuration, and the box geometry, making automated procedures problematic. We present here a general algorithm, named FixBox, that can fix a molecular complex of interest from a minimal set of definitions of its assembling parts and intended arrangement in the simulation box. It uses a unified triclinic framework for the box geometric periodicity, does not require a full molecular topology, and is applicable to various types of systems and configurations, making it possible to fully and easily automate the fixing of a broken molecular complex. The performance of the algorithm is illustrated with problematic configurations of various types of simulated systems. The presented formal framework can generally be useful for algorithms that need to perform geometrical transformations on systems with PBCs.
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